A number of different types of oligonucleotides (and reasonably short polynucleotides) have been described for use in the targeted sequence alteration of DNA, including (i) internally duplexed chimeric RNA-DNA oligonucleotides that fold into a double-stranded, double hairpin conformation, (ii) bifunctional oligonucleotides that include a triplexing domain tethered to a repair domain, and (iii) chemically modified, single-stranded oligonucleotides that have an internally unduplexed DNA correction domain and lack both hairpins and triplexing domains. Various of these oligonucleotides have been shown to effect targeted alteration of single base pairs as well as to introduce frameshift alterations in cells and cell-free extracts from a variety of host organisms, including bacteria, fungi, plants, and animals.
The influence of factors such as growth phase, developmental state, cell cycle position, and the contribution of particular cellular proteins to the efficiency of oligonucleotide-mediated nucleic acid sequence alteration, in either cells or cell-free extracts, is not well understood. Although several cellular pathways and gene groups are known to be involved in mediating in vivo repair of DNA lesions resulting from radiation or chemical mutagenesis (including the RAD52 epistasis group of proteins, the mismatch repair group of proteins, and the nucleotide excision repair group of proteins), and although the role of these proteins in homologous recombination and maintaining genome integrity has been extensively studied (reviewed, for example, in Heyer, Experientia 50(3), 223-233 (1994); Thacker, Trends in Genetics 15(5), 166-168 (1999); Paques & Haber, Microbiol. and Molec. Biol. Rev. 63(2), 349-404 (1999); and Thompson & Schild, Mutation Res. 477, 131-153 (2001)), the specific function of these and related proteins in oligonucleotide-directed nucleic acid sequence alteration is not well understood.
Inhibitors of histone deacetylase (HDAC) induce cultured tumor cells to undergo growth arrest, differentiation, and/or apoptosis. Marks et al., J. Natl. Canc. Inst. 92(15), 1210-1216 (2000). For example, treatment with trichostatin A (TSA), an antibiotic from Streptomyces, results in inhibition of enzymatic activity of partially purified HDAC and accumulation of acetylated histones in various cell types, and can cause induction of Friend cell differentiation and specific inhibition of the cell cycle of normal rat fibroblasts in the G1 and G2 phases at very low concentrations. Yoshida et al., J. Biol. Chem. 265, 17174-17179 (1990).
HDAC inhibitors have also been suggested to affect gene therapy agents. WO 00/23567 discloses methods of promoting stem cell self-renewal that include exposure of a population of stem cells, particularly hematopoietic stem cells, to an effective dose of an HDAC inhibitor, particularly trichostatin A, trapoxin, or chlamydocin. In one embodiment, at least one transgene, either homologous or heterologous to the origin of the recipient DNA, is introduced using retroviral mediated transfer into cells treated with an HDAC inhibitor. In another embodiment, stem cells are genetically modified using a polynucleotide and treatment with an HDAC inhibitor.
WO 00/51424 discloses methods of homologous recombination in cultured non-embryonic stem cells for use as nuclear donors to produce genetically modified animals. The technique was used to insert genes, e.g., a marker gene and a transgene, at different loci using 5′ and 3′ regions that contain between 1.8 and 12 kb of homology at the flanking regions of an insert locus in the chromosome. Agents inhibiting histone deacetylation or factors otherwise stimulating transcription at the target locus are suggested to enhance this homologous recombination process.
WO 00/24917 discloses modification of cellular DNA in vertebrate cells by homologous pairing at preselected locations using parvoviral vectors, including vectors based on adeno-associated virus (AAV). The vectors of this technique, all of which are at least 2.7 kb in length, include a DNA sequence that is substantially identical to a target locus and all or part of at least one parvoviral inverted terminal repeated (ITR) sequence or equivalent. Among the agents disclosed to treat target cells are histone deacetylase inhibitors, such as sodium butyrate and trichostatin A.
HDAC inhibitors have not, however, been suggested or disclosed to increase the efficiency of oligonucleotide-mediated nucleic acid sequence alteration.
Recombination by bacteriophage lambda in E. coli bacteria during lambda's lytic cycle is mediated by the so-called “Red” recombination pathway which comprises two genes. Redo encodes an exonuclease (exo) that binds to the broken ends of double-stranded DNA and degrades one of the strands in the 5′ to 3′ direction, leaving a 3′ single-stranded overhang. Redβ encodes a single-stranded DNA binding protein (bet) that, in combination with the bacterial RecA protein, melts duplex DNA at a site containing sequence complementary to the exposed 3′ end and promotes strand invasion and annealing of the single-strand overhang into the complementary duplex. Red recombination is facilitated by the lambda protein called “Gam” which inhibits the bacterial RecBCD exonuclease, an enzyme that degrades duplex DNA with exposed ends.
Various references describe the use of the Red recombination system to mediate or facilitate homologous recombination in E. coli of linear double stranded DNA of non-lambda phage origin. K. C. Murphy, “Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli,” J. Bacteriol., 180(8):2063-2071 (Apr. 1998); Yu et al., “An efficient recombination system for chromosome engineering in Escherichia coli,” Proc. Natl. Acad. Sci. USA, 97(11):5978-5983 (2000); Ellis et al., “High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides,” Proc. Natl. Acad. Sci. USA, 98(12):6742-6746 (2001).
WO 02/14495 discloses methods for cloning DNA molecules and altering eukaryotic genes in cells having DNA encoding beta protein under the control of a derepressible promoter. The induced beta protein promotes homologous recombination between nucleic acids in the cell, which nucleic acids may be intrachromosomal or extrachromosomal. This publication also discloses methods for inducing homologous recombination using single-stranded DNA molecules by introducing into a cell DNA capable of undergoing homologous recombination and beta protein. The application further discloses bacterial cells that promote efficient homologous recombination, which bacteria contain one or more genes from a defective lambda prophage. However, the this publication states that at least one of the experiments used to describe the invention did not work.
Collectively, the references and international patent publication demonstrate that lambda Red gene products, and in particular beta protein, can be used in bacteria to efficiently alter DNA sequences by homologous recombination using double-stranded and single-stranded oligonucleotides. However, the references neither demonstrate nor suggest that DNA can be altered efficiently using single- or double-stranded oligonucleotides by mechanisms other than homologous recombination, and do not suggest that lambda phage proteins can be used to increase the efficiency of nucleic acid sequence alteration in non-bacterial cells by any mechanism.
Hydroxyurea (HU) is known to inhibit the M2 subunit of ribonucleotide reductase, depleting dNTP pools and impairing DNA replication, Zhou et al., Cancer Res. 55:1328-1333 (1995), which causes cells to arrest at the G1/S border of the cell cycle. HU's ability to inhibit DNA replication has lead to its use as an antiretroviral and as an antineoplastic agent. Hanft et al., Blood 95:3589-3593 (2000); Arbiser et al., Endocrinology 128:972-978 (1991); Tamura et al., J. Investig. Med. 45:160-167 (1997); Lisziewicz, U.S. Pat. No. 6,130,089. HU's ability to arrest the cell cycle at the G1/S checkpoint has been exploited to synchronize cultures of cells prior to genetic manipulations. Hadlaczky et al., WO 97/40183. HU has been shown to stimulate the expression of fetal hemoglobin and has been used to treat sickle cell disease. Steinberg & Rodgers, Medicine (Baltimore) 80:328-344 (2001).
HU has been used to increase the efficiency of retroviral-mediated gene transfer into hematopoietic stem cells. Retroviral integration is most efficient in actively cycling cells. The efficiency of this retroviral transduction is enhanced by the presence of HU in the growth medium used to prepare the target cells. See, e.g., Uchida et al., U.S. Pat. No. 5,928,638. It is believed that the effect of HU is due to its ability to switch quiescent, G0 phase, cells into the more active G1/S/G2 and M phases, giving a population enriched in actively cycling cells.
HU also has been used with adeno-associated virus (AAV) vectors. Alexander et al., U.S. Pat. No. 5,834,182. Like retroviral vectors, AAV vectors act by stably integrating into target cell's chromosome. Tal, J., J. Biomed. Sci. 7:279-291 (2000). As with retroviral transduction, AAV transduction is reported to be more efficient when target cells are pre-treated with HU.
HU has been used to increase the efficiency of nuclear transfer in transgenesis approaches in which cultured cells are first targeted by homologous recombination, and the altered nucleus than transferred. HU is used to synchronize cells prior to donor nucleus isolation, increasing the efficiency of the nuclear transfer process. Colman et al., WO 00/51424.
HU has not, however, been suggested or disclosed to be useful in increasing the efficiency of oligonucleotide-mediated nucleic acid sequence alteration.
A need exists in the art for methods, compositions, and kits to enhance the efficiency of oligonucleotide-mediated nucleic acid sequence alteration, particularly nucleic acid sequence alteration effected by other than homologous recombination. There particularly exists a need in the art for methods, compositions, and kits that can be used to increase the efficiency of oligonucleotide-mediated nucleic acid sequence alteration in eukaryotic cells, such as yeast and mammalian cells, and particularly human cells.